Provided herein is label free electrical methods of detection and characterization of nucleic acid amplification products by probing the dielectric properties of the molecules in solution in a microfluidic device with an interdigitated electrode array.
Currently, the state of the art micro-fabricated devices for polymerase chain reaction (PCR) detection focus mainly on optical detection approach, such as on-chip capillary electrophoresis detection or labeling the amplified products with an intercalating fluorescent dye and reporter particles for detection. Although, those methods are very efficient in comparison to conventional gel electrophoresis methods, the requirement of an optical component integrated into micro-fabricated devices not only limits portability, but also increases the labor and total cost of those devices. Instead of using fluorescence label for optical detection, the electrical properties of DNA molecules can be a good candidate for developing a non-optical PCR detection method.
DNA molecules when suspended in solution will have counter ions surrounding them. These counter ions have two layers; one is associated with the bounded ions, i.e. condensed ions, and the other one is a loosely surrounded ion cloud. When the DNA molecules are probed in an electrical field, the DNA molecules will have longitudinal and transverse dipole moments, formed by counter ions and negatively charged backbone phosphate group.
Most of the research and detection methods of DNA dielectric properties in the past few decades focus on the range of frequency lower than kilohertz, where longitudinal dipole moments dominates the response. However, most of the applications utilizing the longitudinal dipole moments as a means of detection mechanism have not been successful due to complex influences from different types of background ions, charge neutralization and binding to the back-bone, mixed signals from longitudinal and transverse dipole moments, and also length-dependent alpha relaxation.
Instead, by probing at an appropriate frequency range (kilo-Hertz to mega-Hertz), and fitting the measured impedance versus frequency characteristics to an equivalent circuit model, the transverse dipole moment of DNA molecules can be explicitly extracted from mixed dipole moments (longitudinal and transverse) and can be shown to impact the solution dielectric capacitance. This method can also avoid the interference of ionic interactions of DNA solution with a measurement device, such as double layer perturbation, and electrode polarization.
The method, when performed in an integrated manner, can provide point-of-care label-free electronic detection of PCR reactions and nucleic acids in general for a wide range of applications in clinical diagnostics, global health, and individualized medicine.